The functional role of gamma-delta T cells during mycobacterial infection is presently unclear. The bovine calf is an exceptional model for examining the in vivo and in vitro responses of gamma-delta T cells during experimental mycobacterial infection, as neonatal ruminants have large numbers of circulating gamma-delta T cells which comprise as much as much as 75% of the circulating T cell pool. Preliminary data from a bovine model suggests a substantial role for gamma-delta T cells in macrophage activation and bactericidal activity during early infection with Mycobacterium avium subsp. paratuberculosis (Map). The specific aims of this proposed research are to demonstrate that during early Map infection (1) gamma-delta T cells are a predominant T cell subset to proliferate and produce IFN-gamma, and (2) antigen-specific gamma-delta T cells are capable of activating infected macrophages. Preliminary characterization of the immune response to subcutaneous inoculation of Map in this bovine model suggests such a response by antigen-specific gamma-delta T cells from the lymph node draining the site of infection at post- inoculation day (PID) 60. Bacteria are cleared from the skin at the inoculation site and the draining lymph nodes in these animals by PID 60, suggesting that gamma-delta T cells are capable of upregulating macrophage bactericidal mechanisms. Though not ordinarily prominent within lymph nodes, gamma-delta T cells from the lymph node draining the site of infection in half of the animals in preliminary experiments proliferated more extensively and produced more IFN-gammma in response to stimulation with Map- PPD in vitro than either CD4+ or CD8+ alpha-beta T cells from the same lymph node. The functional role of gamma-delta T cells in the modulation of protective immunity to mycobacterial infection has not been established as beneficial, redundant, or deleterious. Understanding the functional role of gamma-delta T cells in mycobacterial infection may be an important consideration in designing immunostimulatory components of future vaccines, and the candidate seeks support to continue development of this animal model for application in human mycobacterial vaccine research. A DVM with research experience in bovine mycobacterial infection, Dr. Simutis is currently engaged in a combined residency/PhD program in veterinary pathology, with strong interest in developing animal models of human diseases.